Zidovudine (3'-azido-3'-deoxythymidine) is one of three nucleoside analogs approved for the treatment of AIDS and AIDS-related complex (Boucher et al., J. Pediatr. 122:137-144 (1993); Fischl et al., N. Engl. J. Med. 323:1009-1014 (1990); Fischl et al., N. Engl. J. Med. 317:185-190 (1987); Lambert et al., N. Engl., J. Med. 322:1333-1340 (1990); Meng et al., Amer. J. Med. 88 (Supp. 15B):27S-30S (1990); Skowron et al., Ann. Inter. Med. 118:321-330 (1993); Yarchoan et al., The Lancet 1:575-580 (1986)).
Investigations of the mode of action of zidovudine have shown that the drug is phosphorylated to its 5'-mono-, di-, and triphosphate derivatives by cellular kinases. Zidovudine-5'-triphosphate is a potent inhibitor of human immunodeficiency virus (herein "HIV") reverse transcriptase and thus of HIV replication (Furman et al., Proc. Natl. Acad. Sci. USA 83:8333-8337 (1986); Mitsuya et al., Proc. Natl. Acad. Sci. USA 82:7096-7100 (1985)). It is also known that zidovudine triphosphate causes reverse transcriptase chain termination (Izuta et al., Biochem. Biophys. Res. Comm. 179 (2):776-783 (1991)).
Much of what is presently known about the intracellular metabolism of zidovudine has been elucidated using radiolabeled drugs in cultured human lymphoid cells. For example, marked differences in the activation and accumulation of zidovudine nucleotides have been noted among different cells upon incubation with zidovudine which correlate with differences in the in vitro effectiveness of the drug (Balzarini et al., Adv. Exp. Med. Biol. 253B:407-413 (1990); Balzarini et al., J. Biol. Chem. 264:6127-6133 (1989)).
Quantitation of zidovudine metabolites has been performed with in vitro cell systems using radiolabeled zidovudine (Furman et al., Proc. Natl. Acad. Sci. USA 83:8333-8337 (1986); Mitsuya et al., Proc. Natl. Acad. Sci. USA 82:7096-7100 (1985)). These studies on the mechanism of action of zidovudine have shown that zidovudine is phosphorylated to its mono-, di-, and triphosphate forms via thymidine kinase and other cellular kinases. Zidovudine triphosphate, as the active form of the drug, is directly responsible for inhibition of reverse transcriptase which ultimately results in inhibition of viral replication by zidovudine (Lambert et al., N. Engl., J. Med. 322:1333-1340 (1990)). However, determination of zidovudine triphosphate in human clinical studies has proven difficult because the use of radiolabeled zidovudine is not feasible in patients.
Since in vitro results from human cell lines in culture cannot necessarily be extrapolated to the in vivo situation and because differences in drug disposition and clinical effects exist in patients, it is of interest to measure the intracellular level of zidovudine triphosphate, the major active metabolite of the drug. More importantly, the evaluation of the intracellular metabolism and pharmacology of the proximate inhibitor of zidovudine would lead to a better understanding of the pharmacological properties of zidovudine in vivo than can obtained from measurement of plasma pharmacokinetics of zidovudine alone.
Two methods have been described for quantitative determination of intracellular zidovudine metabolites in HIV infected patients. One method involves using a multidimensional high pressure liquid chromatography (herein "HPLC") method but its application to measuring cellular zidovudine nucleotides in patients undergoing therapy has not been evaluated (Toyoshima et al., Anal. Biochem. 196:302-307 (1991)). The other method is an indirect assay which utilizes HPLC and radioimmunoassay (herein "RIA"). However, this method is quite cumbersome and requires prior purification of zidovudine metabolites from cell extracts, treatment of extracts with phosphatases and quantitation of the resultant zidovudine with an RIA (Kuster et al., J. Infect. Dis. 164:773-776 (1991); Slusher et al., Antimicrob. Agents Chemother. 36: 2473-2477 (1992)).
Further, in vitro reverse transcription assays are known whereby the inhibitory effects of zidovudine triphosphate on the polymerization of DNA by reverse transcriptase are detected (Lacey et al., J. Biochem. 267 (22): 15789-15794 (1992); Ma et al., J. Med. Chem. 35 (11):1938-1941 (1992); Reardon, J. E., Biochem. 31 (18):4473-4479 ( 1992); Izuta et al., Biochem. Biophys. Res. Comm. 179 (2):776-783 (1991); Parker et al., J. Biochem. 266 (3):1754-1762 (1991); Vrang et al., Antiviral Res. 7 (3):139-149 (1987); Olsen et al., Int. Conf. AIDS 8 (2):PA45 (1992) (Abs. No. PoA 2255); Gronowitz et al., Int. Conf. AIDS 7 (2):113 (1991) (Abs. No. WA1086); White et al., Int. Conf. AIDS 6 (1):186 (Abs. No. ThA266) (1990); White et al., Antiviral Res. (Netherlands) 16/3:257-266 (1991); Furman et al., Am. J. Med. (USA) 85/2 A:176-181 (1988); Vogt et al., Int. Conf. AIDS 5:555 (1989) (Abs. No. Mcp83)). However, the use of reverse transcriptase assays for measuring bodily levels of zidovudine triphosphate have not been reported.
Herein, we describe a simple and convenient bioassay based on the inhibition of reverse transcriptase activity to measure body levels of reverse transcriptase inhibitors, therapeutic compounds and their metabolites, and particularly to quantify intracellular levels of zidovudine triphosphate in cell extracts. Many bodily tissues and cells may be utilized and analyzed in the methods of the invention. The general reproducibility, simplicity and reliability of the method of the invention have been examined and compared favorably to previously studied procedures.